The lepidopteran family Noctuidae includes some of the most destructive agricultural pests, such as the genera Heliothis, Helicoverpa, Spodoptera, and Trichoplusia. For example, included in this family are the tobacco budworm (Heliothis virescens), the cotton bollworm (Helicoverpa zea), the cotton leafworm (Alabama argillacea), the spotted cutworm (Amathes niarum), the glassy cutworm (Crymodes devastator), the bronzed cutworm (Nephelodes emmedonia), the fall armyworm (Laphygma frugiperda), the beet armyworm (Spodoptera exigua), and the variegated cutworm (Peridroma saucia).
Resistance of agricultural pests, such as the Noctuidae (and others), to pesticides leads to environmental and human health risks. This problem of insecticide resistance leads to the use of more non-selective and toxic compounds, in order to overcome pest resistance. This creates a destructive and vicious cycle.
Selective natural toxins have been suggested for use in insect control. These toxins include substances which are produced in specialized glandular tissues in the body of a venomous animal. The venom may be introduced into the body of its prey or opponent, such as by the aid of a stinging-piercing apparatus, in order to paralyze and/or kill it, although other means of delivering venom are known. Scorpions, for example, contain in their venom a number of proteins, or neurotoxins, which are toxic and act on the excitable systems. Among the insect specific toxins suggested for use in insect control are toxins from Bacillus thuringiensis from the scorpions Buthus eupeus and Androctonus australis, Leiurus quinqustriatus hebraeus, Leiurus quinqustriatus quinqustriatus, and from the mite Pyemotes tritici.
The venoms derived from scorpions belonging to the Buthinae subfamily have four main groups of polypeptide neurotoxins which modify axonal sodium conductance. One group of scorpion neurotoxins are the .alpha.-toxins, which selectively affect mammals through an extreme prolongation of the action potentials due to a slowing or blockage of the sodium channel inactivation (Catterall, Science, 223:653-661 (1984); Rochat et al., Advances in Cytopharmacology, pp. 325-334 (1979)). The second group of toxins, the .beta.-toxins, affect sodium channel activation (Couraud and Jover in Handbook of Natural Toxins (Tu, A. Ed.) Vol. 2, pp. 659-678 (1984) New York: Marcel Dekker. The third group of neurotoxins are the depressant insect selective toxins which induce a progressively developing flaccid paralysis of insects by the blockage of action potentials substantially due to the suppression of sodium current (Lester et al., Biochim. Biophys. Acta, 701:370-381 (1982); Zlotkin et al., Arch. Biochem. Biophys., 240:877-887 (1985)). The fourth group of neurotoxins are the excitatory insect selective toxins which cause an immediate (knock down) spastic paralysis of insects by the induction of repetitive firing in their motor nerves due to an increase of the sodium peak current and the voltage dependent slowing of its inactivation (Walther et al., J. Insect Physiol., 22:1187-1194 (1976); Pelhate et al., J. Physiol., 30:318-319 (1981)).
In addition to scorpion and mite toxins, other insect-selective toxins have been identified in venoms from snails, spiders, and a number of other arthropods. [See review by Zlotkin, Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 10, Chapter 15, pp. 499-541 (1985).] The venoms of braconid wasps are highly toxic to lepidopterous larvae. The venom of the braconid Bracon hebetor causes a flaccid paralysis in lepidopterous larvae by inducing presynaptic interruption of the excitatory glutaminergic transmission at the insect neuromuscular junction (Piek et al., Comp. Biochem. Physiol., 72C:303-309 (1982)). The venoms of solitary wasps are toxic to a large number of insects and spiders from different orders (Rathmeyer, Z. Vergl. Physiol., 45:453-462 (1962)). An example of these venoms is the venom of Philanthus triangulum which induces in insects a flaccid paralysis substantially due to presynaptic blockage of neuromuscular transmission; this venom affects both excitatory and inhibitory transmission (May et al., Insect Physiol., 25:285-691 (1979)). The venom of the black widow spider, Latrodectus mactans, contains components which are neurotoxic to insects, but not to mammals, and other components with the opposite selectivity (Fritz et al., Nature, 238:486-487 (1980); Ornberg et al., Toxicon, 14:329-333 (1976)).
More recently, a toxin designated as Lqh.alpha.IT, which strongly reassembles .alpha. toxins in its primary structure and electrophysiological effects, was isolated from the venom L. quinquestriatus hebraeus and was shown to affect mainly insects (Eitan et al., Biochemistry, 29 (1990), pp. 5941-5947).
The venom of venomous animals is composed of a variety of toxins affecting different target sites in the excitable systems of the prey. On the basis of the data comparing the activity of toxins and their respective crude venom towards lepidopterous larvae it is clear that the potency of the crude venom cannot be explained by the activity of one toxin alone. The higher potency of the crude venom could be related to a cooperativity among different toxins in the venom affecting different target sites on the same ionic channels (Table 3, Trainer et al., JBC, 268, 17114-17119 (1993)), different ionic channels on the same excitable cells (Olivera et al., Science, 249, 257-263 (1990)), and/or different binding sites on adjacent excitable cells (nerves and/or muscles) (Olivera et al., Science, 249, 257-263 (1990)).
The depressant and the excitatory insect-selective toxins do not compete with the .alpha. insect toxin for its binding site (Gordon and Zlotkin, FEBS Lett., 315 (1993), pp. 125-128). In contrast to locust or cockroach neuronal membranes the excitatory toxins do not displace the depressant toxins from their binding sites in neuronal membranes of lepidopterous larvae (Gordon et al., Biochemisty, 31 (1992), pp. 76-22-7628; Moskowitz et al., Insect Biochem. Molec. Biol., 24 (1994), pp. 13-19).
Recently, the nuclear polyhedrosis virus Autographa californica (AcNPV), from the family Baculoviridae, has been genetically modified for an increased speed of kill by expressing insect-selective toxins. The introduction of an insect-selective toxin into an insect-pathogenic virus has resulted in a reduction in the killing time of insect hosts, as is described by U.S. Ser. No. 08/229,417, filed Apr. 15, 1994, which is a continuation-in-part application of U.S. Ser. No. 07/629,603, filed Dec. 19, 1990, having (in part) common assignment herewith.
Tomalski et al., U.S. Pat. No. 5,266,317, issued Nov. 30, 1993, discuss use of recombinant baculoviruses that express an insect-specific paralytic neurotoxin of an insect predacious mite. Barton et al., U.S. Pat. No. 5,177,308, issued Jan. 5, 1993, take a different approach in creating transgenic plants that express a scorpion derived insect-specific toxin and/or a soil dwelling microorganism toxin. In a copending application, of common assignment herewith, Hammock and McCutchen, Ser. No. 08/279,956, filed Jul. 5, 1994, discuss insect control with a synergistic combination of recombinant virus and an organic insecticide.
These newly emerging tools using recombinant strategies to control insect pest populations hold promise particularly since the wide-scale presence of pest resistance to organic insecticides, such as pyrethroids, has begun to result in substantial crop losses. In cotton alone, the presence of pyr-R Heliothis species has begun to result in millions of lost dollars annually. In fact, in several cases pyrethroid insecticides have completely failed to control infestations of Heliothis larvae in cotton, which has resulted in complete destruction of the crop.